Biosensor for detection of small molecule analytes

ABSTRACT

Biosensors fur use in detecting analytes, particularly small analytes such as those having a molecular weight of less than 5,000 Daltons, are disclosed which comprise a membrane and an electrode and a reservoir defined therebetween, the membrane having an inner layer proximate the electrode and an outer layer remote from the electrode comprising a closely packed array of amphiphilic molecules, a plurality or ionophores, and a plurality of membrane spanning lipids, the ionophores comprising first and second half membrane spanning monomers, the first half membrane spanning monomers being provided in the inner layer and being prevented from lateral diffusion within the membrane and the second half membrane spanning monomers being provided in the outer layer and being free to diffuse laterally within the membrane, the second half membrane spanning monomers having attached thereto a first receptor which is reactive with the small analyte, wherein a carrier to which is attached a plurality of the analyte is reversibly bound to the first receptor via the analyte. In an alternative embodiment, the second half membrane spanning monomers are attached through a carrier and/or linker group to at least one analyte, and a receptor is reversibly bound to the second half membrane spanning monomers via the analyte and said carrier and/or linker group.

The present invention relates generally to mechanisms for modifying theelectrical conductivity of ionophore containing membrane basedbiosensors in response to the concentration of small analytes or haptensin a sample.

Biomembranes have been constructed from a double layer of closely packedamphiphilic lipid molecules. The molecules of these bilayers exhibit therandom motions characteristic of the liquid phase, in which thehydrocarbon tails of the lipid molecules have sufficient mobility toprovide a soft, flexible, viscid surface. The molecules can also diffusein two dimensions freely within their own monolayer so that twoneighbouring lipids in the same monolayer exchange places with eachother in a time interval several orders of magnitude less than the timefor lipid molecules in opposite monolayers to exchange places.

These membrane may incorporate a class of molecules, called ionophores,which facilitate the transport of ions across these membranes. Ionchannels are a particular form of ionophore, which as the term implies,are channels through which ions may pass through membranes. A favouredionophore is gramicidin A which forms aqueous channels in the membrane.Australian Patent Specification No. 40123/85 discloses the use ofmembranes including ionophores in biosensors. Further examples of gatedionophores are found in Australian Patent Specification No. 21279/88,which discloses receptor molecules conjugated with a support that isremote from the receptor site. The support may be a lipid head group, ahydrocarbon chain, a cross-linkable molecule or a membrane protein ormembrane polypeptide. The inner level of the membrane may be adjacent toa solid surface with groups reactive with the solid surface, and spacedfrom the surface to provide a reservoir region as disclosed inInternational Patent Specification No. 92/17788 (the disclosure of whichis incorporated herein by reference).

Biosensors based on ion channels or ionophores contained within lipidmembranes tethered to or deposited onto metal electrodes are disclosedin Australian Patent Specification Nos. 50334/90 and 40787/89. Thosereferences disclose a membrane bilayer in which each layer hasincorporated therein ionophores and in which the conductance of themembrane is dependent upon the presence or absence of an analyte. Thedisclosure of Australian Patent Specification No. 50334/94 (incorporatedherein by reference) describes various ionophore gating mechanisms tomodify the conductivity of the membrane in response to the presence ofan analyte. In each of those gating mechanisms an inner layer of themembrane (the layer closer to the solid electrode surface, if any)contains immobilised or tethered half membrane spanning ion channelswhile an outer layer contains more mobile half membrane spanning ionchannels. One method for immobilising the ion channels or the innerlayer is to employ a polymerizable lipid layer and then cross-link themolecules of the inner monolayer and the ionophore. The conductivity ofthe membrane is altered by the extent to which opposing half membranespanning ion channels align to establish a membrane spanning channel forion transmission across the membrane.

Other biosensors based on ion channels or ionophores contained withinmembranes are described in International Patent Specification Nos.

WO92/17788, WO93/21528, WO94/07593 and U.S. Pat. No. 5,204,239 (thedisclosures of which are incorporated herein by reference). Theseapplications also disclose methods of producing membranes with improvedsensitivity using a surface amplifier effect, stability and ion fluxusing chemisorbed arrays of amphiphilic molecules attached to anelectrode surface and means of producing lipid membranes incorporatingionophores on said chemisorbed amphiphilic molecules.

The present inventors have now developed a modified biosensor for use inthe detection of small analytes. The modified biosensor can, however,also be used with larger size analytes.

Accordingly in the first aspect the present invention consists in abiosensor for use in detecting analytes, the biosensor comprising amembrane and an electrode and a reservoir defined there between, themembrane having an inner layer proximate the electrode and an outerlayer remote from the electrode comprising a closely packed array ofamphiphilic molecules, a plurality of ionophores, and a plurality ofmembrane spanning lipids, the ionophores comprising first and secondhalf membrane spanning monomers, the first half membrane spanningmonomers being provided in the inner layer and being prevented fromlateral diffusion within the membrane and the second half membranespanning monomers being provided in the outer layer and being free todiffuse laterally within the membrane, the second half membrane spanningmonomers having attached thereto a first receptor which is reactive withthe analyte, wherein a carrier to which is attached a plurality of theanalyte is reversibly bound to the first receptor via the analyte.

In a preferred embodiment of the first aspect of the present invention,a second receptor which is also reactive with the analyte is provided onthe membrane spanning lipids which are prevented from lateral diffusionin the membrane.

The first and second receptors may be the same or different.

The carrier is preferably bound, reversibly, to two or more firstreceptors or two or more first and second receptors, such that thecarrier forms a “bridge” between the membrane members to which thereceptors are attached. In this way, a portion of the second halfmembrane spanning monomers may be caused to locate into a position outof registration with the first half membrane spanning monomers, therebyreducing the conductivity of the membrane.

Preferably, the carrier is substantially larger than the analyte, forexample 2-50 times the molecular weight of the analyte.

In a second aspect the present invention consists in a biosensor for usein detecting analytes, the biosensor comprising a membrane and anelectrode and a reservoir defined there between, the membrane having aninner layer proximate the electrode and an outer layer remote from theelectrode comprising a closely packed array of amphiphilic molecules, aplurality of ionophores, and a plurality of membrane spanning lipids,the ionophores comprising first and second half membrane spanningmonomers, the first half membrane spanning monomers being provided inthe inner layer and being prevented from lateral diffusion within themembrane and the second half membrane spanning monomers being providedin the outer layer and being free to diffuse laterally within themembrane, the second half membrane spanning monomers having attachedthrough a carrier and/or linker group at least one analyte, wherein areceptor which is reactive with the analyte is reversibly bound to thesecond half membrane spanning monomers via the analyte and said carrierand/or linker group.

In a preferred embodiment the receptor has two or more analyte bindingsites thereby allowing the receptor to form a bridge between two or moresecond half membrane spanning monomers. In this way, a portion of thesecond half membrane spanning monomers may be caused to aggregate, outof registration with the first half membrane spanning monomers, therebyreducing the conductivity of the membrane.

Preferably, the analyte binding sites on the receptor are separated byless than 80% of the distance between the first half membrane spanningmonomers. Also, preferably, the second half membrane spanning monomerhave attached through a carrier and/or linker group a plurality of theanalyte.

In a further preferred embodiment of the present invention, the firstreceptors or, in the case of a biosensor according to the second aspect,the analyte or carrier is attached to the second half membrane spanningmonomers via linker groups. Similarly, it is preferred that any secondreceptors are attached to the membrane spanning lipids via linkergroups. Examples of suitable linker groups include protein(s) andpolymers. Preferred linker groups are streptavidin, neutravidin andavidin which may be used to link biotin groups provided on the receptorsand membrane members.

In a third aspect, the present invention consists in a biosensor for usein detecting analytes, the biosensor comprising a membrane and anelectrode and a reservoir defined there between, the membrane having aninner layer proximate the electrode and an outer layer remote from theelectrode comprising a closely packed array of amphiphilic molecules, aplurality of ionophores, and a plurality of membrane spanning lipids,the ionophores comprising first and second half membrane spanningmonomers, the first half membrane spanning monomers being provided inthe inner layer and being prevented from lateral diffusion within themembrane and the second half membrane spanning monomers being providedin the outer layer and being free to diffuse laterally within themembrane, the second half membrane spanning monomers being attachedthrough a linker group to the analyte, wherein a receptor which has twoor more analyte binding sites is reversibly bound to the second halfmembrane spanning monomers via the analyte, and wherein the membranespanning lipids are prevented from lateral diffusion in the membrane andalso have attached the analyte via a linker group.

Thus, in biosensors according to the third aspect, the receptor is ableto bind to analyte attached to second half membrane spanning monomers aswell as analyte attached to the membrane spanning lipids. In this way, aportion of the second half membrane spanning monomers may be caused tolocate to a position out of registration with the first half membranespanning monomers, thereby reducing the conductivity of the membrane.

In order that the nature of these aspects of the present invention maybe more readily understood, preferred forms thereof are shownschematically in FIGS. 1b- 1 f, 2 and 3.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

FIG. 1 schematically depicts various small analytes gating mechanismsfor biosensors: (a) direct gating mechanism; (b) outer channelaggregation; (c) outer/inner channel lateral segregation employing freeantibody; (d) outer/inner channel lateral segregation employing tetheredantibody on ion channel or tethered membrane spanning lipid; (e)outer/inner channel lateral segregation employing tethered antibody onion channel and membrane spanning lipid: and (f) depicts a preferredembodiment of the mechanism shown in (e).

FIGS. 2(a)-(d) depict alternative methods hapten attachment to membranespecies.

FIG. 3 depicts a method of gating by polymerisation.

FIG. 4 provides the chemical structure of linker lipid A which may beused to construct the inner layer of the biosensor membrane.

FIG. 5 provides the chemical structure of linker gramicidin B that maybe used as the immobilised inner layer ion channel in biosensors of thepresent invention.

FIG. 6 provides the chemical structures of membrane spanning lipid C andD. These lipids are preferred examples of lipids suitable forconstructing the inner layer of the biosensor membrane.

FIG. 7 provides a representation of biotinylated gramicidin E.

FIG. 8 depicts the chemical structures for various digoxigeninderivatives.

FIG. 9 provides a spectrograph obtained from a BSA-digoxigenin conjugateusing MALDI (Matrix Assisted Laser Desorption Ionisation) MassSpectroscopy.

FIG. 10 provides a graph of ELISA assay results showing theimmunocompetency of a BSA-digoxigenin conjugate immobilised to amicrotitre plate.

In previous patents and patent applications, mechanisms have beendisclosed for producing an electrical signal, dependent on theconcentration of an analyte, in a biosensor based on an ion-channelcontaining membrane. As described above, the present invention consistsin additional methods for producing such a signal, particularly in thecase where the analyte is a small molecule (say, molecular weight<5000Daltons).

Mechanisms

1. Direct Gating (FIG. 1a)

A receptor, comprising the small analyte of interest, or a derivative oranalogue thereof, is attached to the mobile, outer ion channels. Anantibody, a fragment thereof or another molecular species capable ofrecognising and binding to the small analyte of interest, is then boundto the receptor inducing a change in the ionic conductivity of thechannel. When the above assembly is challenged with a medium containingthe analyte of interest, the analyte competes for the antigen bindingsites of the antibody or other analyte recognising species. In so doinga portion of the antibody or other analyte recognising species, which isrelated to the concentration of analyte in the medium being tested, isremoved from the ion channel-bound receptor, returning it to its initialconducting state.

2. Outer Channel Aggregation (FIG. 1b)

A membrane is prepared with ion channels bearing receptors as describedin Part 1 above. An antibody, a fragment thereof or another molecularspecies capable of recognising and binding to more than one copy of thesmall analyte of interest, is then bound to the receptor. The distancebetween the antigen binding sites of the antibody, or like species, mustbe less than (not more than 80%) the distance between the immobilisedinner layer ion channels. Binding of the antibody, or like species,results in an aggregation of the outer half-membrane-spanningion-channels, out of registration with the inner half-membrane-spanningion channels, reducing the conductivity of the membrane. When the aboveassembly is challenged with a medium containing the analyte of interest,the analyte competes for the antigen binding sites of the antibody orother analyte recognising species. In so doing a portion of the antibodyor other analyte recognising species, which is related to theconcentration of analyte in the medium being tested, is removed from theion channel-bound receptor, returning it to its initial conductingstate.

3. Outer/Inner Channel Lateral Segregation—Free Antibody (FIG. 1c)

A membrane is prepared with ion channels and membrane spanning lipidsbearing receptors as described in Part 1 above. Typically, theconcentration of receptor-bearing membrane spanning lipids in themembrane is much higher than that of the outer ion channels.Furthermore, the concentrations of the membrane spanning lipids and theinner ion channels are chosen such that close location of a membranespanning lipid and an inner ion channel is a rare event. An antibody, afragment thereof or another molecular species capable of recognising andbinding to more than one copy of the small analyte of interest, is thenbound to the receptors. Binding of the antibody, or like species,results in cross-linking the outer ion channels to the immobilisedmembrane spanning lipids, out of registration with the innerhalf-membrane-spanning ion channels, reducing the conductivity of themembrane. When the above assembly is challenged with a medium containingthe analyte of interest, the analyte competes for the antigen bindingsites of the antibody or other analyte recognising species. In so doinga portion of the antibody or other analyte recognising species, which isrelated to the concentration of analyte in the medium being tested, isremoved from the ion channel-bound receptor, returning it to its initialconducting state.

4. Outer/Inner Channel Lateral Segregation—Tethered Antibody on IonChannel or Membrane Spanning Lipid (FIG. 1d)

A membrane is prepared with outer ion channels and membrane spanninglipids in which either the outer ion channels or membrane spanninglipids bear receptors as described in Part 1 above. An antibody, afragment thereof or another molecular species capable of recognising andbinding to the small analyte of interest, is bound to the component ofouter membrane layer (ion channel or membrane spanning lipid) which doesnot bear the receptor. This results in cross-linking of the outer ionchannels to the immobilised membrane spanning lipids, out ofregistration with the inner half-membrane-spanning ion channels,reducing the conductivity of the membrane. When the above assembly ischallenged with a medium containing the analyte of interest, the analytecompetes for the antigen binding sites of the antibody or other analyterecognising species. In so doing a portion of the outer ion channels aredisconnected from the tethered membrane spanning lipids, allowing themto form membrane-spanning ion channels with the innerhalf-membrane-spanning ion channels and increasing the conductivity ofthe membrane.

5. Outer/Inner Channel Lateral Segregation—Tethered Antibody on IonChannel and Membrane Spanning Lipid (FIG. 1e)

A membrane is prepared with outer ion channels and membrane spanninglipids in which antibodies, fragments thereof or other molecular speciescapable of recognising and binding to the small analyte of interest, arebound to the outer ion channel and membrane spanning lipid. The membraneis then treated with a molecular species (e.g. protein, polymer or smallorganic molecule) bearing more than one copy of the small analyte ofinterest, or a derivative or analogue thereof. This results incross-linking of the outer ion channels to the immobilised membranespanning lipids, out of registration with the innerhalf-membrane-spanning ion channels, reducing the conductivity of themembrane. When the above assembly is challenged with a medium containingthe analyte of interest, the analyte competes for the antigen bindingsites of the antibody or other analyte recognising species. In so doinga portion of the outer ion channels are disconnected from the tetheredmembrane spanning lipids, allowing them to form membrane-spanning ionchannels with the inner half-membrane-spanning ion channels andincreasing the conductivity of the membrane.

In preferred embodiment of this gating mechanism, the outerhalf-membrane-spanning ion channels are biotinylated derivates ofgramicidin and the molecular species capable of recognising and bindingto the small analyte of interest is a biotinylated antibody Fab)fragment and is bound to the outer ion channels and to the biotinylatedmembrane spaning lipids via streptavidin, avidin or one of theirmodified analogues. The preferred molecular species (e.g. protein,polymer or small organic molecule) bearing more than one copy of thesmall analyte of interest, or a derivative or analogue thereof is theprotein BSA (bovine serum albumin) functionalised with analyte analoguesattached via amide bonds to BSA lysine side chain amines.

Methods of Attachment of the Receptor to the Membrane Components

In the above examples, a small-analyte-like or antibody-like receptormust be attached to either outer ion channels, membrane spanning lipidsor both. Modes of attachment for the receptor include any of those wellknown to the art, but particularly:

covalent attachment of a single copy of the receptor to the membranecomponent (ion channel or membrane spanning lipid).

covalent attachment of multiple copies of the receptor to the membranecomponent (FIG. 2d), including a covalent attachment of copies of thereceptor to an intermediate species such as a protein (e.g. BSA), orpolymer which is itself attached to the membrane component.

attachment of single (FIG. 2a) or multiple copies (FIG. 2c) of thereceptor to the membrane component via a non-covalent linker e.g.providing biotin groups on the membrane component and receptor which arejoined via a streptavidin or avidin molecule. As would be readilyappreciated by one skilled in the art, any non-covalently associatingpair of sufficient durability could be used in place of streptavidin andbiotin in this role e.g. dinitrophenyl (DNP) hapten and anti-DNPantibody.

covalent attachment of the receptor to a species such as streptavidinwhich can then be non-covalently bound to the membrane component (FIG.2b).

It should be noted that, in the cases where multiple copies of thesmall-analyte-like receptor are attached to a single outer ion channel,the possibility exists of gating by polymerisation of the ion channelson addition of an antibody-like species with two or more binding sties(FIG. 3). This represents an extended version of gating by outer channelaggregation (FIG. 1b).

EXAMPLE Method for Construction of the Membrane

The supported bilayer for use in small analyte sensing using multimerichapten species may be constructed as described in the co-pendinginternational (PCT) application entitled “Self-Assembly of SensorMembranes” filed Jun. 20, 1996.

That is, an electrode coated with a clean gold surface is contacted witha solution containing linker lipid A (FIG. 4), the disulfide ofmercaptoacetic acid (maad), linker gramicidin B (FIG. 5), membranespanning lipid C (FIG. 6) and membrane spanning lipid D, the disulfidecontaining components in the solution thus adsorbing onto the goldsurface of the electrode. Typically the solution may contain thedisulfides in the following concentrations:

Linker lipid A 1    mM maad 1    mM Linker gramicidin B 0.0001 mMMembrane spanning lipid C 0.0010 mM Membrane spanning lipid D 0.0001 mMdissolved in ethanol

The electrode is then rinsed and the excess organic solvent used forrinsing is removed. The second layer of the lipid bilayer is then formedby adding a solution of lipid and biotinylated gramicidin E (FIG. 7),dispersed in a suitable solvent onto the electrode surface containing afirst layer and rinsing the electrode surface with an aqueous solution.Typically the solution to form the second layer might contain:

Diphytanylphosphatidylcholine 7    mM Glyceryl diphytanyl ether 3    mMand biotinylated gramicidin E 0.0002 mM in ethanol

To the bilayer membrane thus formed is added a solution of streptavidin,avidin, neutravidin or other avidin or streptavidin derivative(typically at a 50 μg/ml concentration for a 10 minute incubation). Theelectrode is then rinsed with aqueous solution in order to remove excessstreptavidin, avidin, neutravidin or other avidin or streptavidinderivative, and a solution of a biotinylated receptor molecule is added(eg a biotinylated anti-hapten antibody fragment).The membrane is thenrinsed and a solution containing the multi-hapten carrying species isadded (typically at a concentration of 10-100 nM for 10-15 minutes). Themembrane is then given a final rinse.

Requirements for the “Poly hapten Species” (i.e. Carrier Having Attacheda Plurality of Analyte)

In the minimalist case, the multihapten species can be a dimer of haptenlinked by a short carbon (or other) carrier chain (say, 4-50 atoms inlength). Alternately, a carrier such as a protein (eg bovine serumalbumin or polylysine), polysaccharide (e.g. dextran or carboxymethyldextran) or synthetic polymer (e.g. polyacrylic acid) or copolymer canbe used. It is envisaged that the use of proteins or polymers will beadvantageous for providing additional solubility to haptens of sparingsolubility in aqueous media. A molecular weight for a protein or polymerin the range of 10,000 to 100,000 Daltons would be expected to beoptimal.

Preparation of Multi-Hapten Bovine Serum Albumin Conjugate

Preparation of Digoxigenin NHS ester (3)

(Digoxigenin derivatives illustrated in FIG. 8)

Digoxigenin-3-hemisuccinate was prepared by the method detailed in U.S.Pat. No. 3,855,208. This hemisuccinate (70 mg, 0.1426 mmol),N-hydroxysuccimimide (NHS) (98 mg, 6 equivalents),dicyclohexylcarbodiimide (DCC) (118 mg, 4 equivalents) andN,N-dimethyl-4-aminopyridine (DMAP) (18 mg, 1 equivalent) were dissolvedin tetrahydrofuran (THF) (10 ml) and stirred under an atmosphere ofnitrogen, overnight at room temperature.

The mixture was filtered to remove precipitated dicyclohexylurea (DCU).The filtrate was evaporated and added toN-(6-aminocaproyl)-6-aminocaproic acid benzyl ester (XXBn) (95 mg, 2equivalents) at pH 8 in 25% MeOH/DCM (5 ml). The reaction mixture wasstirred at room temperature overnight.

The mixture was evaporated and purified by silica gel chromatography ona 2×10 cm column (eluent 5% MeOH/DCM). Fractions 26-45 (5 ml fractions)contained the product and were evaporated and dried. The residue waswashed with water, and dried to give (1) (68 mg).

A solution of (1) (58 mg) in MeOH (5 ml) was stirred with a catalyticamount of palladium-on-charcoal under a hydrogen atmosphere for 1 hour.The mixture was then filtered and evaporated to give the free acid (2)(50 mg).

A solution of (2) (50 mg, 0.069 mmol), DCC (43 mg, 3 equivalents) andNHS (9 mg, 5 equivalents) in dry, distilled THF (10 ml) was stirred atroom temperature overnight under a nitrogen atmosphere. The mixturefiltered to remove DCU and evaporated to give digoxigenin NHS ester (3).This material was used without further purification.

Synthesis of Digoxigenin Conjugates

A solution of digoxigenin NHS ester (3) in ethanol (20 mM, 4ml, 0.08mmol) was added to a stirred solution of bovine serum albumin (BSA) (250mg. 0.0037 mmol) dissolved in 0.05 M sodium phosphate (10 ml, pH 8.0)and 10 ml dimethylformamide (DMF). After 3 days incubation, the reactionwas dialyzed in 0.05 M sodium phosphate buffer for 24 hours. The proteinconcentration was estimated by absorbance at 280 nm and the conjugatewas stored at 4° C. The average number of haptens coupled to each BSAmolecule was estimated by MALDI (Matrix Assisted Laser DesorptionIonisation) Mass Spectroscopy to be 6 digoxigenins per BSA (FIG. 9). Theimmunocompetency of the digoxigenin-BSA conjugate was demonstrated byELISA assay, with the BSA-digoxigenin conjugate immobilised on themicrotitre plate (FIG. 10).

DETECTION ANALYTE

The detection of analyte is achieved by addition of an analytecontaining sample to the membrane assembly prepared as described above.The analyte in the sample competes with the analyte/analyte analogues onthe carrier for the receptors attached to the membrane species,disconnecting the bridge between the ion channels and membrane spanninglipids. The consequence is an increase in membrane conductivity which isrelated to the analyte concentration in the sample.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

What is claimed is:
 1. A biosensor for use in detecting analytes, thebiosensor comprising a membrane and an electrode and a reservoir definedthere between, the membrane having an inner layer proximate theelectrode and an outer layer remote from the electrode comprising aclosely packed array of amphiphilic molecules, a plurality ofionophores, and a plurality of membrane spanning lipids, the ionophorescomprising first and second half membrane spanning monomers, the firsthalf membrane spanning monomers being provided in the inner layer andbeing prevented from lateral diffusion within the membrane and thesecond half membrane spanning monomers being provided in the outer layerand being free to diffuse laterally within the membrane, the second halfmembrane spanning monomers having attached thereto a first receptorwhich is reactive with the analyte, wherein a carrier to which isattached a plurality of the analyte is reversibly bound to the firstreceptor via the analyte, said analyte having a molecule weight lessthan 5,000 Daltons.
 2. A biosensor according to claim 1, wherein thefirst receptors are attached to the second half membrane spanningmonomers via linker groups.
 3. A biosensor according to claim 2, whereinthe linker groups comprise streptavidin, neutravidin or avidin.
 4. Abiosensor according to claim 1, wherein the carrier is bound,reversibly, to two or more first receptors such that the carrier forms abridge between the second half membrane spanning monomers to which thefirst receptors are attached.
 5. A biosensor according to claim 1,wherein a second receptor which is also reactive with the analyte isprovided on the membrane spanning lipids which are prevented fromlateral diffusion in the membrane.
 6. A biosensor according to claim 1,wherein the second receptors are attached to the membrane spanninglipids via linker groups.
 7. A biosensor according to claim 1, whereinthe carrier is bound, reversibly, to at least one first and secondreceptors such that the carrier forms a bridge between the membranemembers to which the first and second receptors are attached.
 8. Abiosensor according to claim 1, wherein the carrier is substantiallylarger than the analyte.
 9. A biosensor according to claim 1, whereinthe carrier is a protein, polysaccharide or polymer.
 10. A biosensoraccording to claim 9, wherein the carrier is selected from bovine serumalbumin, polylysine, dextran, carboxymethyldextran or polyacrylic acid.11. A biosensor according to claim 9, wherein the carrier has amolecular weight which is 2-50 times larger than that of the analyte.12. A biosensor according to claim 9, wherein the carrier has amolecular weight in the range of 10,000 to 100,000 Daltons.
 13. Abiosensor according to claim 1, wherein the receptor is an antibody. 14.A biosensor according to claim 1, wherein the second half membranespanning monomers are gramicidin A or amphotericin B monomers.
 15. Abiosensor according to claim 1, wherein the analyte is digoxigenin. 16.A biosensor for use in detecting analytes, the biosensor comprising amembrane and an electrode and a reservoir defined there between, themembrane having an inner layer proximate the electrode and an outerlayer remote from the electrode comprising a closely packed array ofamphiphilic molecules, a plurality of ionophores, and a plurality ofmembrane spanning monomers, the ionophores comprising first and secondhalf membrane spanning monomers, the first half membrane spanningmonomers being provided in the inner layer and being prevented fromlateral diffusion within the membrane and the second half membranespanning monomers being provided in the outer layer and being free todiffuse laterally within the membrane, the second half membrane spanningmonomers having attached through a carrier and/or linker group at leastone analyte, wherein a receptor which is reactive with the analyte isreversibly bound to the second half membrane spanning monomers via theanalyte and said carrier and/or linker group, said analyte having amolecule weight less than 5,000 Daltons.
 17. A biosensor according toclaim 16, wherein the receptor has two or mote analyte binding sitessuch that the receptor forms a bridge between two or more second halfmembrane spanning monomers.
 18. A biosensor according to claim 17,wherein the analyte binding sites on the receptor are separated by lessthan 80% of the distance between the first half membrane spanningmonomers.
 19. A biosensor according to claim 16, wherein the second halfmembrane spanning monomers are attached through a carrier and/or linkergroup to a plurality of analyte.
 20. A biosensor according to claim 19,wherein the plurality of the analytes is present on a carrier which isattached to the second half membrane spanning monomers.
 21. A biosensoraccording to claim 20, wherein the carriers are attached to the secondhalf membrane spanning monomers via linker groups.
 22. A biosensoraccording to claim 16, wherein the analyte(s) are attached to the secondhalf membrane spanning monomers via linker groups.
 23. A biosensoraccording to claim 16, wherein the carrier is a protein, polysaccharideor polymer.
 24. A biosensor according to claim 23, wherein the carrieris selected from bovine serum albumin, polylysine, dextran,carboxymethyldextran or polyacrylic acid.
 25. A biosensor according toclaim 23, wherein the carrier has a molecular weight which is 2-50 timeslarger than that of the analyte.
 26. A biosensor according to claim 23,wherein the carrier has a molecular weight in the range of 10,000 to100,000 Daltons.
 27. A biosensor according to claim 16, wherein thefirst half membrane spanning monomers are gramicidin E.
 28. A biosensoraccording to claim 16, wherein the carrier is a protein, polysaccharideor polymer.
 29. A biosensor according to claim 28, wherein the carrieris selected from bovine serum albumin, polylysine, dextran,carboxymethyldextran or polyacrylic acid.
 30. A biosensor according toclaim 28, wherein the carrier has a molecular weight which is 2-50 timeslarger than that of the analyte.
 31. A biosensor according to claim 28,wherein the carrier has a molecular weight in the range of 10,000 to100,000 Daltons.
 32. A biosensor according to claim 16, wherein thereceptor in an antibody.
 33. A biosensor according to claim 16, whereinthe linker groups comprise streptavidin, neutravidin or avidin.
 34. Abiosensor according to claim 16, wherein the second half membranespanning monomers are gramicidin A or amphotericin B monomers.
 35. Abiosensor according to claim 16, wherein the first half membranespanning monomers are gramicidin E.
 36. A biosensor according to claim16, wherein the analyte is digoxigenin.
 37. A biosensor for use indetecting analytes, the biosensor comprising a membrane and an electrodeand a reservoir defined there between, the membrane having an innerlayer proximate the electrode and an outer layer remote from theelectrode comprising a closely packed array of amphiphilic molecules, aplurality of ionophores, and a plurality of membrane spanning lipidscomprising first and second half membrane spanning monomers, the firsthalf membrane spanning monomers being provided in the inner layer andbeing prevented from lateral diffusion within the membrane and thesecond half membrane spanning monomers being provided in the outer layerand being free to diffuse laterally within the membrane, the second halfmembrane spanning monomers being attached through a linker group to theanalyte, wherein a receptor which has two or mote analyte binding sitesis reversibly bound to the second half membrane spanning monomers viathe analyte, and wherein the membrane spanning lipids are prevented fromlateral diffusion in the membrane and also have attached the analyte viaa linker group, said analyte having a molecule weight less than 5,000Daltons.
 38. A biosensor according to claim 37, wherein the receptor isan antibody.
 39. A biosensor according to claim 37, wherein the linkergroups comprise streptavidin, neutravidin or avidin.
 40. A biosensoraccording to claim 37, wherein the second half membrane spanningmonomers are gramicidin A or amphotericin B monomers.
 41. A biosensoraccording to claim 37, wherein the first half membrane spanning monomersare gramicidin E.
 42. A biosensor according to claim 37, wherein theanalyte is digoxigenin.